To study plasticity, we cultured cortical networks on multielectrode arrays, enabling simultaneous recording from multiple neurons. We used conditional firing probabilities to describe functional network connections by their strength and latency. These are abstract representations of neuronal pathways and may arise from direct pathways between two neurons or from a common input. Functional connections based on direct pathways should reflect synaptic properties. Therefore, we searched for long-term potentiation (this mechanism occurs in vivo when presynaptic action potentials precede postsynaptic ones with interspike intervals up to ∼20 ms) in vitro. To investigate if the strength of functional connections showed a similar latency-related development, we selected periods of monotonously increasing or decreasing strength. We observed increased incidence of short latencies (5-30 ms) during strengthening, whereas these rarely occurred during weakening. Furthermore, we saw an increased incidence of 40-65 ms latencies during weakening. Conversely, functional connections tended to strengthen in periods with short latency, whereas strengthening was significantly less than average during long latency. Our data suggest that functional connections contain information about synaptic connections, that conditional firing probability analysis is sensitive enough to detect it and that a substantial fraction of all functional connections is based on direct pathways. © 2009 by the Biophysical Society.